The invention pertains to the therapeutic use of non-neurotoxic plasminogen activators especially from the saliva of Desmodus rotundus (DSPA) preferentially for the treatment of stroke.
Different clinical pictures are summarized under the term “stroke” which correlate in their clinical symptoms. According to the respective pathogenesis a first differentiation between these clinical pictures in so called ischaemic and haemorrhagic insults is possible.
Ischaemic insults (ischaemia) are characterized in a reduction or interruption of the blood circulation in the brain due to a lack of arterial blood supply. Often this is caused by thrombosis of an arteriosclerotic stenosed vessel or by arterio arterial, respecitively, cardial embolisms.
Haemorrhagic insults are based inter alia on the perforation of brain supplying arterias damaged by arterial hypertonia. However, only approximately 20% of all cerebral insults are caused by haemorrhagic insults. Thus, stroke due to thrombosis is much more relevant.
In comparison to other tissue ischaemias the ischaemia of the neuronal tissue is widely accompanied by necrosis of the effected cells. The higher incidence of necrosis in neuronal tissue can be explained with the new understanding of the phenomenon “excitotoxicity” which is a complex cascade comprising a plurality of reaction steps. The cascade is initiated by ischaemic neurons affected by a lack of oxygen which then lose ATP instantaneously and depolarize. This results in an increased postsynaptic release of the neurotransmitter glutamate which activates membrane bound glutamate receptors regulating cation channels. However, due to the increased glutamate release glutamate receptors become over activated.
Glutamate receptors regulate voltage dependent cation channels which are opened by a binding of glutamate to the receptor. This results in a Na+ and Ca2+ influx into the cell massively disturbing the Ca2+ dependent cellular metabolism. Especially the activation of the Ca2+ dependent catabolic enzymes could give reason to the subsequent cell death (Lee, Jin-Mo et al., “The changing landscape of ischaemic brain injury mechanisms”; Dennis W. Zhol “Glutamate neurotoxicity and diseases of the nervous system”).
Although the mechanism of glutamate mediated neurotoxicity is not yet entirely understood it is agreed upon that it contributes in a large extent to the neuronal cell death following cerebral ischaemia (Jin-Mo Lee, et al.).
Besides safeguarding vital functions and stabilizing physiological parameter the reopening of the closed vessel has priority in the therapy of acute cerebral ischaemia. The reopening can be performed by different means. The mere mechanical reopening, as e.g. the PTCA after heart attack, so far has not yet led to satisfying results. Only with a successful fibrinolysis an acceptable improvement of the physical condition of patients can be achieved. This can be accomplished by a local application using a catheter (PROCAT, a study with prourokinase). However, despite first positive results this method has not yet been officially approved as a pharmaceutical treatment.
The naturally occurring fibrinolysis is based on the proteolytic activity of the serine protease plasmin which originates from its inactive precursor by catalysis (activation). The natural activation of plasminogen is catalyzed by the plasminogen activators u-PA (urokinase type plasminogen activator) and t-PA (tissue plasminogen activator) occurring naturally in the body. In contrast to u-PA, t-PA forms a so called activator complex together with fibrin and plasminogen. Thus, the catalytic activity of t-PA is fibrin dependent and is enhanced in its presence approximately 550-fold. Besides fibrin also fibrinogen can stimulate t-PA mediated catalysis of plasminogen to plasmin—even though to a smaller extent. In the presence of fibrinogen the t-PA activity is only increases 25-fold. Also the cleavage products of fibrin (fibrin degradation products (FDP)) are stimulating t-PA.
Early attempts of thrombolytic treatment of acute stroke go back to the 1950s. First extensive clinical trials with streptokinase, a fibrinolytic agent from beta-haemolysing streptococci, started only in 1995. Together with plasminogen streptokinase forms a complex which catalyzes other plasminogen molecules into plasmin.
The therapy with streptokinase has severe disadvantages since it is a bacterial protease and therefore can provoke allergic reactions in the body. Furthermore, due to a former streptococci infection including a production of antibodies the patient may exhibit a so called streptokinase resistance making the therapy more difficult. Besides this, clinical trials in Europe (Multicenter Acute Stroke Trial of Europe (MAST-E), Multicenter Acute Stroke Trial of Italy (MAST-1)) and Australia (Australian Streptokinase Trial (AS7)) indicated an increased mortality risk and a higher risk of intracerebral bleeding (intracerebral haemorrhage, ICH) after treating patients with streptokinase. These trials had to be terminated early.
Alternatively, urokinase—also a classical fibrinolytic agent—can be applicated. In contrast to streptokinase it does not exhibit antigenic characteristics since it is an enzyme naturally occurring in various body tissues. It is an activator of plasminogen and independent of a cofactor. Urokinase is produced in kidney cell cultures.
Extensive experience on therapeutic thrombolysis is available for the tissue type plasminogen activator—the so called rt-PA—(see EP 0 093 619, U.S. Pat. No. 4,766,075), which is produced in recombinant hamster cells. In the 90s several clinical trials were performed world-wide using t-PA—with acute myocardial infarction as the main indication—leading to partially non-understood and contradictory results. In the so called European Acute Stroke Trial (ECASS) patients were treated within a time frame of 6 hours after the onset of the symptoms of a stroke intravenously with rt-PA. After 90 days the mortality rate as well as the Barthel-index were examined as an index for the disability or the independent viability of patients. No significant improvement of the viability was reported but an—even though not significant—increase of mortality. Thus, it could be concluded, a thrombolytic treatment with rt-PA of patients being individually selected according to their respective case history immediately after the beginning of the stroke could possibly be advantageous. However, a general use of rt-PA within the time frame of 6 hours after the onset of stroke was not recommended since an application during this time increases the risk of intracerebal haemorrhage (ICH) (C. Lewandowski C and Wiliam Barsan, 2001: Treatment of Acute Stroke; in: Annals of Emergency Medicine 37:2; S. 202 ff.).
The thrombolytic treatment of stroke was also subject of a clinical trial conducted by the National Institute of Neurologic Disorder and Stroke (so called NINDS rtPA Stroke Trial) in the USA. This trial concentrated on the effect of intravenous rt-PA treatment within only three hours after the onset of the symptoms. Patients were examined three months after the treatment. Due to the observed positive effects of this treatment on the viability of patients, rt-PA treatment within these limited time frame of three hours was recommended although the authors found a higher risk for ICH.
Two further studies (ECASS II Trial: Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischaemic Stroke (ATLANTIS)) examined whether the positive effects of rt-PA treatment within three hours after the onset of stroke could be repeated even with a treatment within six hours time. However, this question could not be answered affirmatively since no improvement of the clinical symptoms or any decrease in mortality was observed. The higher risk for ICH remained.
Those partially contradictory results have led to a high caution in the use of rt-PA. Already 1996 a publication of the American Heart Association pointed out the strong skepticism among doctors with respect to thrombolytic treatment of stroke; whereas there is no such skepticism with respect to fibrinolytica in the therapy of myocardical infarct (van Gijn J. MD, FRCP, 1996-Circulation 1996, 93: 1616-1617).
A rational behind this skepticism was firstly given in a summary of all stroke trials published 1997 (updated in March 2001). According to this review all thromtolytica treatments (urokinase, streptokinase, rt-PA or recombinant urokinase) resulted in a significant higher mortality within the first 10 days after the stroke while the total number of either dead or disabled patents was reduced when the thrombolytica where applied within six hour after stroke onset. This effects were mainly due to ICH. The broad use of thrombolytica for the treatment of stroke was therefore not recommended.
Even before, such results gave reason to some other authors mere sarcastic statement that stroke patients had the choice to either die or to survive disabled (SCRIP 1997: 2265, 26).
Nevertheless, so far the therapy with rt-PA is the only treatment of acute cerebral ischaemia approved by the Food and Drug Administration (FDA) in the USA. However, it is restricted to an application of rt-PA within three hours after the onset of stroke.
The approval of rt-PA was reached in 1996. Before, in the year 1995, first announcements about negative side effects of t-PA became known, which provide an explanatory basis for its dramatic effects when applied in stroke treatment outside the three hour time frame. Accordingly, micoglia cells and neuronal cells of the hippocampus produce t-PA which contributes to the glutamate mediated excitotoxicity. This is concluded from a comparative study on t-PA deficient and wild type mice when glutamate agonists were injected in their hippocampus, respectively. The t-PA deficient mice showed a significant higher resistance against external (inthrathecal) applicated glutamate (Tsirka S E et al., Nature, Vol. 377, 1995, “Excitoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator”). These results were confirmed in 1998 when Wang et al. could prove nearly a double quantity of necrotic neuronal tissue in t-PA deficient mice when t-PA was injected intravenously. This negative effect of external t-PA on wild type mice was only approximately 33% (Wang et al., 1998, Nature, “Tissue plasminogen activator (t-PA) increases neuronal damage after focal cerebral ischaemia in wild type and t-PA deficient mice”.)
Further results on the stimulation of excitotoxicity by t-PA were published by Nicole et al. In the beginning of 2001 (Nicole O., Docagne F Ali C; Margaill I; Carmeliet P; MacKenzie E T, Vivien D and Buisson A, 2001: The proteolytic activity of tissue plasminogen activator enhances NMDA receptor-mediated signaling; in: Nat Med 7, 59-64). They could prove that t-PA being released by depolarized cortical neurons could interact with the so called NR1 sub-unit of the glutamate receptor of the NMDA type leading to a cleavage of NR1. This increases the receptor's activity resulting in a higher tissue damage after glutamate agonist NMDA was applied. The NMDA agonist induced excitotoxicity.
Thus, t-PA exhibits a neurotoxic effect by activating the glutamate receptor of the NMDA type.
According to a further explanatory concept the neurotoxicity of t-PA results indirectly from the conversion of plasminogen in plasmin. According to this model plasmin is the effector of neurotoxicity (Chen Z L and Strickland S, 1997: Neuronal Death in the hippocampus is promoted by plasmin-catalysed degradation of laminin. Cell; 91, 917-925).
A summarizing outline of the time depending neurotoxic effect of t-PA is given in FIG. 5. Therein also the increased toxicity of the recombinant t-PA compared to endogenic t-PA becomes evident. This is probably due to rt-PA being able to enter into tissue in higher concentrations.
Despite its neurotoxic side effect and its increasing effect on the mortality t-PA was approved by FDA. This can only be explained by the lack of harmless and effective alternatives—thus it is due to a very pragmatic cost benefit analysis. Therefore, there is still a need for safe therapies. However, if they were still based on thrombolytica—in case it is not possible to find alternatives to thrombolysis—the problem of neurotoxicity has to be considered (see for example Wang et al. a.a.O.; Lewandowski and Barson 2001 a.a.O.).
Therefore, further examination of known thrombolytica including DSPA (Desmodus rotundus Plasminogen Activator) in order to develop new drugs for stroke was terminated although principally all thrombolytica are potentially suitable. Especially in case of DSPA its potential suitability for this medical indication was pointed out earlier (Medan P; Tatlisumak T; Takano K; Carano R A D; Hadley S J; Fisher M: Thrombolysis with recombinant Desmodus saliva plasminogen activator (rDSPA) in a rat embolic stroke model; in: Cerebrovasc Dis 1996: 6; 175-194 (4.sup.th International Symposium on Thrombolic Therapy in Acute Ischaemic Stroke), DSPA is a plasminogen activator with a high homology (resemblance) to t-PA. Therefore—and in addition to the disillusionment resulting from the neurotoxic side effects of t-PA—there were no further expectations, for DSPA being a suitable drug for stroke treatment.
Instead, recent strategies aiming to improve known thrombolytic treatments try to apply the thrombolytic substance no longer intravenously but intraarterially via a catheter directly close to the intravascular thrombus. First experience is available with recombinant produced urokinase. Thus, possibly, the necessary dose for thrombolysis and therewith negative side effects could be reduced. However, this application requires a high technical expenditure and is not available everywhere. Furthermore, the patient has to be prepared in a time consuming action. Time, however, is often limited. Thus, the preparation provides for an additional risk.
Presently, new concepts are directed to anticoagulants such as heparin, aspirin or ancrod, which is the active substance in the poison of the Malayan pit viper. Two further dinical trials examining the effects of heparin (International Stroke Trial (IST) and Trial of ORG 10172 in Acute Stroke Treatment (TOAST)) however, do not indicate a significant improvement of mortality or a prevention of stroke.
A further new treatment focuses neither on thrombus nor on blood thinning or anti coagulation but attempts to increase the vitality of cells damaged by the interruption of blood supply (WO 01/51613 A1 and WO 01/51614 A1). To achieve this antibiotics from the group of quinons, aminoglycosides or chloramphenicol are applied. For a, similar reason it is further suggested to begin with the application of citicholin directly after the onset of stroke. In the body, citicholin is cleaved to cytidine and choline. The cleavage products form part of the neuronal cell membrane and thus support the regeneration of damaged tissue (U.S. Pat. No. 5,827,832).
Recent research on safe treatment is based on the new finding that a part of the fatal consequences of stroke is caused only Indirectly by interrupted blood supply but directly to the excito- or neurotoxicity including over activated glutamate receptors. This effect is increased by t-PA (see above). A concept to reduce excitotoxicity is therefore to apply so called neuroprotectives. They can be used separately or in combination with fibrinolytic agents in order to minimize neurotoxic effects. They can lead to a reduced excitotoxicity either directly e.g. as a glutamate receptor antagonist or indirectly by inhibiting voltage dependent sodium or calcium channels (Jin-Mo Lee et al. a.a.O.).
A competitive inhibition (antagonistic action) of the glutamate receptor of NMDA type is possible e.g. with 2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonoheptanoate (APH). A non competitive inhibition can be achieved e.g. by substances binding to the phencyclidine side of the channels. Such substances can be phencyclidine, MK801, dextrorphane or cetamine.
So far, treatments with neuroprotectives have not shown the expected success, possibly because neuroprotectives had to be combined with thrombolytic agents in order to exhibit their protective effects. This applies to other substances (see also FIG. 6).
Even a combination of t-PA and neuroprotective agents results only in a limited damage. Nevertheless, the disadvantageous neurotoxicity of the fibrinolytic agent as such is not avoided.